WO2018183922A1 - Procédés et compositions pour vacciner contre le paludisme - Google Patents

Procédés et compositions pour vacciner contre le paludisme Download PDF

Info

Publication number
WO2018183922A1
WO2018183922A1 PCT/US2018/025510 US2018025510W WO2018183922A1 WO 2018183922 A1 WO2018183922 A1 WO 2018183922A1 US 2018025510 W US2018025510 W US 2018025510W WO 2018183922 A1 WO2018183922 A1 WO 2018183922A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
virus
cells
dna
antigenic
Prior art date
Application number
PCT/US2018/025510
Other languages
English (en)
Inventor
Joao Carlos Aguiar
Keith Limbach
Emily C. SMITH
Original Assignee
The Henry M. Jackson Foundation For The Advancement Of Military Medicine, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Henry M. Jackson Foundation For The Advancement Of Military Medicine, Inc. filed Critical The Henry M. Jackson Foundation For The Advancement Of Military Medicine, Inc.
Priority to US16/497,244 priority Critical patent/US20210260176A1/en
Priority to EP18776071.5A priority patent/EP3600399A4/fr
Publication of WO2018183922A1 publication Critical patent/WO2018183922A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A61P33/06—Antimalarials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0003—Invertebrate antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/002—Protozoa antigens
    • A61K39/015—Hemosporidia antigens, e.g. Plasmodium antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • C07K14/445—Plasmodium
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • a computer readable text file entitled “SequenceListing.text,” created on or about March 27, 2018 with a file size of about 593 KB contains the sequence listing for this application and is hereby incorporated by reference herein in its entirety.
  • the protective T cell response following vaccination with RAS is dominated by CD8+ T cells specific for the major surface protein on the sporozoite, the circumsporozoite protein (CSP), although T cell responses specific for other antigens can also contribute to protection.
  • CSP circumsporozoite protein
  • T cell responses specific for several antigens have been observed following RAS immunization.
  • vaccination with SPZ+CQ allows expression of the full repertoire of liver-stage genes and replication of the parasite in
  • Plasmodium spp. life cycle are particularly promising targets for malaria vaccine development, with great potential to prevent infection and transmission.
  • the pre-erythrocytic stages of the parasitic life cycle are vulnerable to vaccine intervention because their antigens are expressed at a time when low numbers of sporozoites are transmitted by the mosquito to the human host and only a few hepatocytes become infected.
  • the present invention provides methods and compositions for immunizing a subject against malaria, with the methods comprising administering an immunologically effective amount of at least one antigenic polypeptide having an amino acid sequence that is at least 90, 95 or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:ll, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:40, NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:l,
  • FIGURE 1 depicts a schematic view of high-throughput Ad-array generation and antigen identification assays. The general steps involved in the generating a defined array of adenovectors and their use in antigen discovery screens using high-throughput technology are indicated.
  • FIGURE 2 depicts generation of the Ad-array.
  • A >300 highly expressed malaria pre-erythrocytic genes were amplified using P. yoelii genomic DNA and gene-specific primers. The reaction products were electrophoresed on 1% agarose gels with a 1 KB ladder as shown here for a subset of these genes. The control is a pair of oligos used to amplify the El region of Ad5 DNA.
  • B Parallel generation of two Ad-array vectors in multi-well plates.
  • the schematic indicates two pAdFlex plasmids (pAdgPyHepl7 and pAdgCMVp65), which were linearized with Pac I and transfected into 293 cells in 60 mm, 6 well, 12 well, 24, well, 48 well and 96 well plates. Following two passages in 293 cells in the same plate size, CPE was observed in all wells. Viral DNA was obtained, and PCR analysis was performed using primers that flank the expression cassette. The products of the PCR reaction were loaded into a 1% agarose gel and electrophoresed. Arrows next to AdgPyHepl7 and AdgCMVp65 indicate the expected size for the PCR products. Plate sizes used to generate the recombinant adenovectors are indicated.
  • FIGURE 3 depicts that adenovector expressed antigens are effective at recalling T cell responses from immunized mice.
  • A Schema for in vitro antigen discovery.
  • B Ad5 vector effectively transduces APC.A20 cells that were infected with AdGFP at the indicated MOI. The percentage of GFP positive cells was determined by FACS.
  • C AdPyCSP infected APCs can recall CD8+ T cell responses from mice immunized using a PyCSP DNA vaccine.
  • Target A20 cells were infected with various MOI of an Ad5 vector expressing PyCSP (AdPyCSP).
  • FIGURE 4 depicts that adenovector expressed antigens are effective at recalling CD8+ T cell responses from mice immunized with protective regimens of sporozoite vaccines.
  • Target A20 cells were infected with Ad5 vector (either triple CsC purified AdPyCSP or unpurified cell lysate from AdPyCSP infected cells) at the indicated MOI and incubated with splenocytes from RAS immunized mice.
  • Control targets were A20 cells infected with AdNull, AdGFP and uninfected A20 cells, (a) IFNy+ cells were measured by ELISpot.
  • FIGURE 5 depicts the identification of targets of CD8+ T cell responses induced by highly protective SPZ+CQ vaccine regimen.
  • Splenocytes from BALB/c mice immunized with SPZ+CQ were screened for CD8+ recall responses specific for 312 pre-erythrocytic antigens.
  • the mean of the negative controls is indicated by the horizontal line.
  • the dotted line indicates responses that are > 2 SD above the mean of the negative controls.
  • FIGURE 6 depicts that the P. falciparum Ortholog of PY03674, PF3D7_0725100 (SEQ ID NO. is Immunogenic in BALB/c Mice.
  • Mice were immunized with 1 x 10 9 PU of GC46.PF3D7_0725100 or control adenovector that does not express a transgene, GC46.Null.
  • Three weeks post-immunization mice were euthanized and PF3D7_0725100-specific CD8+ (A) and CD4+ (B) T cell responses were measured by intracellular cytokine staining and flow cytometry following 4-hr stimulation using pooled overlapping 15-mer peptides.
  • FIGURE 7 depicts identification of protective and immunogenic antigens using a matrix format.
  • A Antigens (numbered 1-9) are grouped into six pools of three antigens (labeled A-F). Each antigen is present in two pools. For example, Antigen 9 is in both pools C (with 7 and 8) and F (with 3 and 6). Each pool is tested alone, and also in combination with PyCSP; therefore, each antigen is tested in four groups of mice.
  • B and C CDl mice are immunized with DNA (a pool of three antigen-expressing constructs with or without PyCSP) followed by Ad5-boost at six weeks with pooled vectors expressing the corresponding antigens.
  • mice Null-immunized (4X, matching the largest dose) and naive mice are also included as negative controls, and PyCSP alone is included as a positive control.
  • B Two weeks following immunization, mice are challenged with 300 infectious P. yoelii sporozoites. Sterile protection is assessed by blood smear.
  • C Two weeks following immunization, mice are challenged with 10,000 infectious P. yoelii sporozoites by intravenous injection. Forty-two hours after challenge, mice are euthanized and livers harvest for immunological analyses and assessment of protection by quantification of liver parasite burden.
  • FIGURE 8 depicts the use of matricies comprised of pooled adenovectors to identify T-cell antigens.
  • Groups of 14 CDl mice were immunized with DNA/HuAd5 vectors expressing groups of P. yoelii antigens, as described in Figure 7. All mice were IV challenged with 300 non-lethal 17XNL P. yoelii sporozoites.
  • the present invention provides methods and compositions for immunizing a subject against malaria, with the methods comprising administering at least one antigenic polypeptide having an amino acid sequence that is at least 90, 95 or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:ll, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:40, NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, NO:51,
  • the present invention provides the use of compositions for immunizing a subject against malaria, with the use comprising administering at least one antigenic polypeptide having an amino acid sequence that is at least 90, 95 or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:ll, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:40, NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, NO:51
  • polypeptides disclosed herein are or possess novel antigens, or are orthologs thereof, that display a positive reaction to at least one of two types of screening assays for antigenicity. It is possible that the polypeptides, antigenic fragments thereof and/or orthologs thereof, also display a positive reaction to additional screening assays for antigenicity.
  • the polypeptides or fragments thereof can promote or provide a positive stimulus in an antigenic screening assay comprising flow cytometry (FACS) identification of lymphocytes stimulated in vitro with splenocytes from vaccinated animals, or the polypeptides or fragments thereof can provide or promote a positive stimulus in an antigenic screening assay comprising Enzyme-linked ImmunoSpot (EliSpot) identification of lymphocytes stimulated in vitro with splenocytes from vaccinated animals.
  • FACS flow cytometry
  • EliSpot Enzyme-linked ImmunoSpot
  • modified parasites containing the polypeptides or fragments thereof are administered to an animal with a spleen, and the splenocytes are subsequently harvested and screened for their ability to stimulate production of antigenic substances, such as but not limited to interferon gamma (IFNy), interleukin-2 (IL-2), from lymphocytes in vitro.
  • antigenic substances such as but not limited to interferon gamma (IFNy), interleukin-2 (IL-2), from lymphocytes in vitro.
  • IFNy interferon gamma
  • IL-2 interleukin-2
  • the invention is directed to polypeptides or fragments thereof that promote a positive in vitro antigenic response in lymphocytes.
  • the phrase "promoting a positive antigenic response" is used herein to mean the polypeptides or fragments thereof can cause production of antigenic substances from lymphocytes, either directly or indirectly, such as using stimulated splenocytes as described above.
  • the polypeptides disclosed herein are novel antigens, or the orthologs thereof, that have also been shown to induce an "antibody response” and/or a "cellular immune response” in mice immunized with radiation-attenuated sporozites ( AS) from Plasmodium yoelii.
  • AS radiation-attenuated sporozites
  • the present invention provides methods of inducing an antibody response in a subject in need thereof comprising administering at least one of the polypeptides or the antigenic fragment thereof to a subject capable of producing an antibody response.
  • the present invention also provides methods of inducing a cellular immune response in a subject in need thereof comprising administering at least one of the polypeptides or the antigenic fragment thereof to the subject.
  • an antibody response is used as it is in the art. Namely, an antibody response occurs when a subject's immune system produces antibodies that bind specifically to an antigen upon being exposed to the antigen.
  • the antibodies may be free in the subject's blood plasma, or the antibodies may be membrane-bound, which are often referred to as "B cell receptors" (BC s).
  • An antibody response as used herein, may include production of free antibodies found in blood, tissue or other body fluids, or the antibody response may include production of membrane-bound antibodies, or both.
  • a cellular immune response or “cell mediated immunity” is an immune response in a subject that does not involve antibodies.
  • a cellular immune response includes activation of specific cell types, such as but not limited to phagocytes, and T cells, as well as release of various cytokines from immune cells.
  • IL-1 interleukin 1
  • IL-6 tumor necrosis factor alpha
  • IFNct interferon alpha
  • IFN beta IFN beta
  • IFNy IFN gamma
  • TGF transforming growth factor beta
  • IL-4 IL-10 and IL-13.
  • protein and “peptide” are used interchangeably and simply used to denote at least a polymer, branched or unbranched, of amino acid residues.
  • isolated when used in conjunction with proteins and nucleic acids, is used to indicate that the proteins or nucleic acids are present in a form in which the protein does not naturally occur.
  • the antigenic proteins of the present application are proteins that naturally occur in P.
  • the isolated antigenic proteins or fragments described herein can be purified or substantially purified.
  • the term "purified” when used in reference to a protein or nucleic acid means that the concentration of the molecule being purified has been increased relative to other molecules associated with it in its natural environment, or environment in which it was produced, found or synthesized.
  • these "other molecules” might include proteins, nucleic acids, lipids and sugars but generally do not include water, solvents, buffers, and reagents added to maintain the integrity or facilitate the purification of the molecule being purified.
  • a substance may be 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100% pure when considered relative to its contaminants.
  • fragment when used in connection with a protein, is used to mean a peptide that contains a sequence of contiguous amino acids taken from the full length or mature antigenic proteins.
  • the antigenic protein fragments of the present invention comprise or alternatively consist of sequences of contiguous amino acids that are about 0.01 to 0.05, 0.1 to 0.5, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, 35 to 40, 40 to 45, 45 to 50, 50 to 55, 55 to 60, 60 to 65, 65 to 70, 70 to 75, 75 to 80, 80 to 85, 85 to 90, 90 to 95 or about 95 to 100 percent of the full length amino acid sequences disclosed herein.
  • the fragments of the antigenic proteins may or may not possess similar antigenicity as the full length antigenic proteins.
  • the fragments of the present invention are antigenic.
  • the fragments of the present invention are immunogenic.
  • the polypeptides of the invention may be immunologically cross-reactive and may be capable of eliciting in an animal an immune response to P. falciparum, P. vivax or P. yoelii, or infected cells thereof or antigen presenting cells expressing P. falciparum or P. yoelii antigens and/or are able to be bound by anti- protein antibodies.
  • antigenic refers to a substance such as a peptide or nucleic acid to which an antibody or T-cell receptor specifically binds.
  • immunogenic refers to a peptides ability to elicit at least a partial cellular immune response or antibody response.
  • the terms “correspond(s) to” and “corresponding to,” as they relate to sequence alignment, are intended to mean enumerated positions within a reference protein, e.g., SEQ ID NO:17, and those positions in a modified protein that align with the positions on the reference protein.
  • a reference protein e.g., SEQ ID NO:17
  • the amino acids in the subject sequence that "correspond to" certain enumerated positions of the reference sequence are those that align with these positions of the reference sequence, but are not necessarily in these exact numerical positions of the reference sequence.
  • amino acid residues of the antigenic proteins of the present invention may or may not be modified such as, but not limited to, addition of functional or non-functional groups such a but not limited to, acetyl groups, hydroxyl groups, carboxyl groups, carbohydrate groups (glycosylation), phosphate groups and lipid groups to name a few. Any of numerous chemical modifications may be carried out by known techniques, including but not limited to, specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH 4 , acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin, etc.
  • the antigenic proteins of the present invention may or may not contain additional elements that, for example, may include but are not limited to regions to facilitate purification.
  • "histidine tags" (“his tags") or "lysine tags” may be appended or “fused” to the antigenic proteins to create “antigenic fusion proteins.”
  • histidine tags include, but are not limited to hexaH, heptaH and hexaHN.
  • lysine tags include, but are not limited to pentaL, heptaL and FLAG.
  • Such regions may be removed prior to final preparation of the antigenic proteins.
  • Other examples of a second fusion peptide include, but are not limited to, glutathione S-transferase (GST) and alkaline phosphatase (AP).
  • peptide moieties to antigenic proteins, whether to engender secretion or excretion, to improve stability and to facilitate purification or translocation, among others, is a familiar and routine technique in the art and may include modifying amino acids at the terminus to
  • the N-terminus amino acid may be modified to, for example, arginine and/or serine to accommodate a tag.
  • the amino acid residues of the C-terminus may also be modified to accommodate tags.
  • One particularly useful fusion protein comprises a heterologous region from immunoglobulin that can be used solubilize proteins.
  • fusion proteins include but are not limited to, fusions with secretion signals and other heterologous functional regions.
  • a region of additional amino acids, particularly charged amino acids may be added to the N-terminus of the antigenic proteins to improve stability and persistence in the host cell, during purification or during subsequent handling and storage.
  • fusion polypeptides of the invention includes an antigenic polypeptide, fragment or variant thereof fused to a polypeptide having adjuvant activity, such as the subunit B of either cholera toxin or E. coli heat labile toxin.
  • a fusion polypeptide encompassed by the invention includes an antigenic polypeptide fused to a cytokine, such as, but not limited to, IL-2, IL-4, IL-10, IL-12, or interferon.
  • An antigenic polypeptide of the invention can be fused to the N- or C-terminal end of a polypeptide having adjuvant activity.
  • an antigenic polypeptide of the invention can be fused within the amino acid sequence of the polypeptide having adjuvant activity.
  • the antigenic polypeptides, and fusions thereof may comprise sequences that form one or more epitopes of a native P. falciparum and/or P. yoelii polypeptides that elicit bactericidal or opsonizing antibodies and/or CD8 + T cells.
  • Such antigenic polypeptides may be identified by their ability to generate antibodies and/or CD8 + T cells that kill cells infected with P.
  • the present invention provides antibodies that specifically bind to one or more of the antigenic peptides of the present invention.
  • isolated or purified preparations of an antigenic polypeptide of the present invention can be used as an immunogen in an immunogenic composition.
  • the same immunogen can be used to immunize mice for the production of hybridoma lines that produce monoclonal antibodies.
  • the antigenic polypeptides of the present invention are used as immunogens.
  • the peptides may be produced by protease digestion, chemical cleavage of isolated or purified polypeptide, chemical synthesis or by recombinant expression, after which they are then isolated or purified.
  • Such isolated or purified peptides can be used directly as immunogens.
  • useful peptide fragments are 8 or more amino acids in length.
  • Useful immunogens may also comprise such peptides conjugated to a carrier molecule, such as a carrier protein.
  • Carrier proteins may be any commonly used in immunology, include, but are not limited to, bovine serum albumin (BSA), chicken albumin, keyhole limpet hemocyanin (KLH), tetanus toxoid, synthetic T cell epitopes and the like.
  • useful immunogens for eliciting antibodies of the invention comprise mixtures of two or more of any of the above-mentioned individual immunogens.
  • Immunization of animals with the immunogens described herein for example in humans, rabbits, rats, ferrets, mice, sheep, goats, cows or horses, can be performed following procedures well known to those skilled in the art, for purposes of obtaining antisera containing polyclonal antibodies or hybridoma lines secreting monoclonal antibodies.
  • Monoclonal antibodies can be prepared by standard techniques, given the teachings contained herein. Such techniques are disclosed, for example, in U.S. Patent No. 4,271,145 and U.S. Patent No. 4,196,265, which are incorporated by reference. Briefly, an animal is immunized with the immunogen. Hybridomas are prepared by fusing spleen cells from the immunized animal with myeloma cells. The fusion products are screened for those producing antibodies that bind to the immunogen. The positive hybridomas clones are isolated, and the monoclonal antibodies are recovered from those clones.
  • Immunization regimens for production of both polyclonal and monoclonal antibodies are well known in the art.
  • the immunogen may be injected by any of a number of routes, including
  • the immunogen may be injected in soluble form, aggregate form, attached to a physical carrier, or mixed with an adjuvant, using methods and materials well known in the art.
  • the antisera and antibodies may be purified using column chromatography methods well known to those of skill in the art.
  • the antibodies of the invention may be used in passive immunization to prevent or attenuate P. falciparum and/or P. yoelii infections of animals, including humans.
  • a cytotoxic antibody is one that enhances opsonization and/or complement killing of the protozoan bound by the antibody.
  • neutralizing antibody is one that reduces the infectivity of the P. falciparum and/or P. yoelii and/or blocks binding of P. falciparum, P. vivax and/or P. yoelii to a target cell.
  • An effective concentration of polyclonal or monoclonal antibodies raised against the immunogens of the invention may be administered to a host to achieve such effects.
  • concentration of the antibodies administered will vary according to each specific antibody preparation, but may be determined using standard techniques well known to those of ordinary skill in the art. Administration of the antibodies may be accomplished using a variety of techniques, including but not limited to those described herein.
  • antibodies is intended to include all forms, such as but not limited to polyclonal, monoclonal, purified IgG, IgM, or IgA antibodies and fragments thereof, including but not limited to antigen binding fragments such as Fv, single chain Fv (scFv), F(ab)2, Fab, and F(ab)' fragments, single chain antibodies as disclosed in U.S. Pat. No. 4,946,778 (incorporated by reference), as well as complementary determining regions (CDR) as disclosed in Verhoeyen and Winter, in Molecular
  • Further aspects of the invention include chimeric and/or humanized antibodies (U.S. Patent Nos. 5,225,539; 5,585,089; and 5,530,101; all of which are incorporated by reference) in which one or more of the antigen binding regions of the antibody is introduced into the framework region of a heterologous (e.g. human) antibody.
  • the chimeric or humanized antibodies of the invention are less antigenic in humans than non-human antibodies but have the desired antigen binding and other activities, including but not limited to neutralizing activity, cytotoxic activity, opsonizing activity or protective activity.
  • the antibodies of the invention are human antibodies.
  • Human antibodies may be isolated, for example, from human immunoglobulin libraries (see, e.g., PCT publications WO 9846645, WO 9850433, WO 9824893, WO 9816054, WO 9634096, WO 9633735, and WO 9110741, all of which are incorporated by reference) by, for example, phage display techniques (see, e.g., PCT publications WO 9002809; WO 9110737; WO 9201047; WO 9218619; WO 9311236; WO 9515982; WO 9520401 and U.S. Patent Nos.
  • Human antibodies may also be generated from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins, see, e.g., U.S. Patent No. 5,939,598, which is incorporated by reference. Human antibodies may also be generated as described in U.S. Patent Application No. 20130291134 which is herein incorporated by reference.
  • the invention also provides polynucleotides that code for the isolated antigenic proteins disclosed herein.
  • the nucleic acids of the invention can be DNA or RNA, for example, mRNA.
  • the nucleic acid molecules can be double-stranded or single-stranded; single stranded RNA or DNA can be the coding, or sense, strand or the non-coding, or antisense, strand.
  • the nucleic acids may encode any of the antigenic proteins disclosed herein, as well as variants thereof.
  • the nucleic acids of the present invention may encode additional elements, such as his tags and the like.
  • the nucleic acids of the invention would include those that encode any of the antigenic proteins and variants thereof that are also contain a glutathione-S-transferase (GST) fusion protein, poly-histidine (e.g., Hiss), poly-HN, poly-lysine, etc.
  • GST glutathione-S-transferase
  • the nucleotide sequences can include additional non-coding sequences such as non-coding 3' and 5' sequences (including regulatory sequences, for example).
  • nucleic acids encoding the antigenic polypeptides of the present invention may be produced by methods well known in the art.
  • nucleic acids encoding the antigenic polypeptides can be derived from polypeptide coding sequences by recombinant DNA methods known in the art.
  • the coding sequence of an antigenic polypeptide may be altered by creating amino acid substitutions that will not affect the immunogenicity of the antigenic polypeptide or which may improve its immunogenicity, such as conservative or semi-conservative substitutions as described above.
  • Various methods may be used, including but not limited to, oligonucleotide directed, site specific mutagenesis. This and other techniques known in the art may be used to create single or multiple mutations, such as replacements, insertions, deletions, and transpositions, for example, as described in Botstein (1985) Science 229:1193-1210 which is incorporated by reference.
  • the identified and isolated DNA encoding the antigenic polypeptides of the present invention can be inserted into an appropriate cloning vector.
  • a large number of vector-host systems known in the art may be used.
  • the term "host” or "host cell” as used herein refers to either in vivo in an animal or in vitro in mammalian cell cultures.
  • the present invention also comprises vectors containing the nucleic acids encoding the antigenic proteins of the present invention.
  • a "vector" may be any of a number of nucleic acids into which a desired sequence may be inserted by restriction and ligation for transport between different genetic environments or for expression in a host cell.
  • Vectors are typically composed of DNA although NA vectors are also available.
  • Vectors include, but are not limited to, plasmids and phagemids.
  • a cloning vector is one which is able to replicate in a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector may be cut in a determinable fashion and into which a desired DNA sequence may be ligated such that the new recombinant vector retains its ability to replicate in the host cell.
  • An expression vector is one into which a desired DNA sequence may be inserted by restriction and ligation such that it is operably joined to regulatory sequences and may be expressed as an RNA transcript.
  • Vectors may further contain one or more marker sequences suitable for use in the identification and selection of cells which have been transformed or transfected with the vector.
  • Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes which encode enzymes whose activities are detectable by standard assays known in the art (e.g., ⁇ - galactosidase or alkaline phosphatase), and genes which visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques.
  • vectors include but are not limited to those capable of autonomous replication and expression of the structural gene products present in the DNA segments to which they are operably joined.
  • the vectors to be used are those for expression of polynucleotides and proteins of the present invention.
  • such vectors comprise cis-acting control regions effective for expression in a host operatively linked to the polynucleotide to be expressed.
  • Appropriate transacting factors are supplied by the host, supplied by a complementing vector or supplied by the vector itself upon introduction into the host.
  • vectors can be used to express the proteins of the invention.
  • Such vectors include chromosomal, episomal and virus-derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements, from viruses such as adeno-associated virus, lentivirus, baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. All may be used for expression in accordance with this aspect of the present invention.
  • any vector suitable to maintain, propagate or the fusion proteins in a host may be used for expression in this regard.
  • compositions comprise an expression vector the contains a nucleic acid that encodes at least one of the proteins of the invention, wherein the DNA expression vector is a DNA plasmid, alphavirus, replicon, adenovirus, poxvirus, adenoassociated virus, cytomegalovirus, canine distemper virus, yellow fever virus, retrovirus, RNA replicons, DNA replicons, alphavirus replicon particles, Venezuelan Equine Encephalitis virus, Semliki Forest Virus or Sindbus Virus.
  • the DNA expression vector is a DNA plasmid, alphavirus, replicon, adenovirus, poxvirus, adenoassociated virus, cytomegalovirus, canine distemper virus, yellow fever virus, retrovirus, RNA replicons, DNA replicons, alphavirus replicon particles, Venezuelan Equine Encephalitis virus, Semliki Forest Virus or Sindbus Virus.
  • the DNA sequence in the expression vector is generally operably linked to appropriate expression control sequence(s) including, for instance, a promoter to direct mRNA transcription.
  • promoters include, but are not limited to, the phage lambda PL promoter, the E. coli lac, trp and tac promoters, HIV promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name just a few of the well-known promoters.
  • expression constructs will contain sites for transcription, initiation and termination and, in the transcribed region, a ribosome binding site for translation.
  • the coding portion of the mature transcripts expressed by the constructs will include a translation initiating AUG at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
  • constructs may contain control regions that regulate, as well as engender expression. Generally, such regions will operate by controlling transcription, such as repressor binding sites and enhancers, among others.
  • Vectors for propagation and expression generally will include selectable markers. Such markers also may be suitable for amplification or the vectors may contain additional markers for this purpose.
  • the expression vectors may contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells.
  • Preferred markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, and tetracycline, kanamycin or ampicillin resistance genes for culturing E. coli and other bacteria.
  • Promoter/enhancer elements which may be used to control expression of inserted sequences include, but are not limited to the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto (1980) Cell 22:787-797), the herpes thymidine kinase promoter (Wagner (1981) Proc. Natl. Acad. Sci. U.S.A.
  • trc for expression in bacterial cells (see also "Useful proteins from recombinant bacteria” in Scientific American, 1980, 242:74-94), the nopaline synthetase promoter region or the cauliflower mosaic virus 35S RNA promoter (Gardner (1981) Nucl. Acids Res.
  • Any method known in the art for inserting DNA fragments into a vector may be used to construct expression vectors containing an antigenic polypeptide encoding nucleic acid molecule comprising appropriate transcriptional/translational control signals and the polypeptide coding sequences. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombination.
  • vectors for expressing heterologous proteins in bacterial hosts include but are not limited to pZE O, pTrc99A, pUC19, pUC18, pKK223-3, pEXl, pCAL, pET, pSPUTK, pTrxFus, pFastBac, pThioHis, pTrcHis, pTrcHis2, and pLEx.
  • the phage in lambda GEMTM-11 may be utilized in making recombinant phage vectors which can be used to transform host cells, such as E. coli LE392.
  • the vector is pQE30 or pBAD/ThioE, which can be used transform host cells, such as E. coli.
  • the invention also provides for host cells comprising the nucleic acids and vectors described herein.
  • host-vector systems may be utilized to express the polypeptide-coding sequence. These include but are not limited to mammalian cell systems infected with virus (e.g., vaccinia virus, adenoviris, etc.); insect cell systems infected with virus (e.g., baculovirus); microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA, plant cells or transgenic plants.
  • virus e.g., vaccinia virus, adenoviris, etc.
  • insect cell systems infected with virus e.g., baculovirus
  • microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA, plant cells or transgenic plants.
  • Hosts that are appropriate for expression of nucleic acid molecules of the present invention, fragments, analogues or variants thereof, may include E. coli, Bacillus species, Haemophilus, fungi, yeast, such as Saccharomyces, Pichia, Bordetella, or Candida, or the baculovirus expression system.
  • a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Expression from certain promoters can be elevated in the presence of certain inducers; thus, expression of the genetically engineered antigenic polypeptides may be controlled.
  • different host cells have characteristic and specific mechanisms for the translational and post-translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed.
  • recombinant expression vectors can be propagated and prepared in quantity.
  • a recombinant polypeptide of the invention is produced and can be recovered in a substantially purified from the cell paste, the cell extract or from the supernatant after centrifugation of the recombinant cell culture using techniques well known in the art.
  • the recombinant polypeptide can be purified by antibody-based affinity purification, preparative gel electrophoresis, or affinity purification using tags (e.g., 6X histidine tag) included in the recombinant polypeptide.
  • the present invention also provides therapeutic and prophylactic compositions, which may be antigenic compositions or immunogenic compositions, including vaccines, for use in the treatment or prevention (reducing the likelihood) of P. falciparum, P. vivax and/or P. yoelii infections in human subjects (patients).
  • the immunogenic compositions include vaccines for use in humans.
  • compositions of the present invention can be prepared by techniques known to those skilled in the art and comprise, for example, an immunologically effective amount of any of the antigenic proteins or fragments thereof, disclosed herein, optionally in combination with or fused to or conjugated to one or more other immunogens, including lipids, phospholipids, carbohydrates, lipopolysaccharides, inactivated or attenuated whole organisms and other proteins, of P. falcipaurm and/or P. yoelii origin or other bacterial origin, a pharmaceutically acceptable carrier, optionally an appropriate adjuvant, and optionally other materials traditionally found in vaccines.
  • immunogens including lipids, phospholipids, carbohydrates, lipopolysaccharides, inactivated or attenuated whole organisms and other proteins, of P. falcipaurm and/or P. yoelii origin or other bacterial origin, a pharmaceutically acceptable carrier, optionally an appropriate adjuvant, and optionally other materials traditionally found in vaccines.
  • the invention provides a cocktail vaccine comprising several antigens, which has the advantage that immunity against one or several strains of a single pathogen or one or several pathogens can be obtained by a single administration.
  • antigens include, but are not limited to, those used in the known DPT vaccines, H MW protein of C. trachomatis or fragments thereof, MOMP of C. trachomatis or fragments thereof, or PMPH or HtrA of C.
  • trachomatis or fragments thereof preferably epitope containing fragments
  • immunogenic amount or "immunologically effective amount” is used herein to mean an amount sufficient to induce an immune response.
  • the immunogenic composition is one that elicits an immune response sufficient to prevent or reduce the likelihood of P. falcipaurm and/or P. yoelii infections or to attenuate the severity of any preexisting or subsequent P. falcipaurm and/or P. yoelii infection.
  • An immunogenic amount of the immunogen to be used in the vaccine is determined by means known in the art in view of the teachings herein. The exact
  • concentration will depend upon the specific immunogen to be administered, but can be determined by using standard techniques well known to those skilled in the art for assaying the development of an immune response.
  • an effective amount of a composition of the invention produces an elevation of antibody titer after administration.
  • approximately 0.01 to 2000 ⁇ g, or 0.1 to 500 ⁇ g, or 50 to 250 ⁇ g of the protein administered is to a host.
  • Compositions which induce CD8 + Tcell responses which are bactericidal or reactive with host cells infected with P. falcipaurm and/or P. yoelii are also an aspect of the invention.
  • Additional compositions comprise at least one adjuvant.
  • the combined immunogen and carrier or diluent may be an aqueous solution, emulsion or suspension or may be a dried preparation.
  • Appropriate carriers are known to those skilled in the art and include stabilizers, diluents, and buffers. Suitable stabilizers include carbohydrates, such as sorbitol, lactose, mannitol, starch, sucrose, dextran, and glucose, and proteins, such as albumin or casein.
  • Suitable diluents include saline, Hanks Balanced Salts, and Ringers solution.
  • Suitable buffers include an alkali metal phosphate, an alkali metal carbonate, or an alkaline earth metal carbonate.
  • the composition of the invention is formulated for administration to humans.
  • compositions, including vaccines, of the invention are prepared by techniques known to those skilled in the art, given the teachings contained herein.
  • an immunogen is mixed with the carrier to form a solution, suspension, or emulsion.
  • One or more of the additives discussed herein may be added in the carrier or may be added subsequently.
  • the vaccine preparations may be desiccated or lyophilized, for example, by freeze drying or spray drying for storage or formulations purposes. They may be subsequently reconstituted into liquid vaccines by the addition of an appropriate liquid carrier or administered in dry formulation using methods known to those skilled in the art, particularly in capsules or tablet forms.
  • Immunogenic, antigenic, pharmaceutical and vaccine compositions may further contain one or more auxiliary substance, such as wetting or emulsifying agents, pH buffering agents, or adjuvants to enhance the effectiveness thereof.
  • Immunogenic, antigenic, pharmaceutical and vaccine compositions may be administered to fish, birds, humans or other mammals, including ruminants, rodents or primates, by a variety of administration routes, including parenterally, intradermal ⁇ , intraperitoneally, subcutaneously or intramuscularly.
  • the immunogenic, antigenic, pharmaceutical and vaccine compositions formed according to the present invention may be formulated and delivered in a manner to evoke an immune response at mucosal surfaces.
  • the immunogenic, antigenic, pharmaceutical and vaccine compositions may be administered to mucosal surfaces by, for example, the nasal, oral (intragastric), ocular, bronchiolar, intravaginal or intrarectal routes.
  • binders and carriers may include, for example, polyalkalene glycols or triglycerides.
  • Oral formulations may include normally employed incipients such as, for example, pharmaceutical grades of saccharine, cellulose and magnesium carbonate.
  • compositions can take the form of microspheres, solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain about 0.001 to 95% of an antigenic protein.
  • Some dosage forms may contain 50 ⁇ g to 250 ⁇ g of an antigenic protein.
  • the immunogenic, antigenic, pharmaceutical and vaccine compositions are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective, protective or immunogenic.
  • the compositions may optionally comprise an adjuvant.
  • the immunogenic, antigenic, pharmaceutical and vaccine compositions may be used in combination with or conjugated to one or more targeting molecules for delivery to specific cells of the immune system and/or mucosal surfaces.
  • targeting molecules include but are not limited to vitamin B12, bacterial toxins or fragments thereof, monoclonal antibodies and other specific targeting lipids, proteins, nucleic acids or carbohydrates.
  • Suitable regimes for initial administration and booster doses are also variable, but may include an initial administration followed by subsequent administrations, such as a booster adminitration.
  • the dose may also depend on the route(s) of administration and will vary according to the size of the host.
  • the concentration of the protein in an antigenic, immunogenic or pharmaceutical composition according to the invention is in general about 0.001 to 95%, specifically about 0.01 to 5%.
  • the antigenic, immunogenic or pharmaceutical preparations, including vaccines may comprise as the immunostimulating material a nucleic acid vector comprising at least a portion of the nucleic acid molecule encoding at least one antigenic protein.
  • a vaccine comprising nucleic acid molecules encoding one or more of the antigenic polypeptides or fragments thereof of the present invention or fusion proteins as described herein, such that the polypeptide is generated in situ is provided.
  • the nucleic acid molecules may be present within any of a variety of delivery systems known to those skilled in the art, including nucleic acid expression systems, bacterial and viral expression systems. Appropriate nucleic acid expression systems contain the necessary nucleotide sequences for expression in the patient such as suitable promoter and terminating signals.
  • the nucleic acid molecules may be introduced using a viral expression system (e.g., vaccinia or other pox virus, alphavirus retrovirus or adenovirus) which may involve the use of nonpathogenic (defective) virus. Techniques for incorporating nucleic acid molecules into such expression systems are well known to those of ordinary skill in the art.
  • the nucleic acid molecules may also be administered as "naked" plasmid vectors as described, for example, in Ulmer (1992) Science 259:1745- 1749. Techniques for incorporating DNA into such vectors are well known to those of ordinary skill in the art.
  • a vector may additionally transfer or incorporate a gene for a selectable marker (to aid in the identification or selection of transduced cells) and/or a targeting moiety, such as a gene that encodes a ligand for a receptor on a specific target cell, to render the vector target specific.
  • a targeting moiety such as a gene that encodes a ligand for a receptor on a specific target cell, to render the vector target specific.
  • Targeting may also be accomplished using an antibody, by methods know to those skilled in the art.
  • Nucleic acid molecules (DNA or NA) of the invention can be administered as vaccines for therapeutic or prophylactic purpose.
  • a DNA molecule is placed under the control of a promoter suitable for expression in a mammalian cell.
  • the promoter can function ubiquitously or tissue- specifically. Examples of non-tissue specific promoters include but are not limited to the early cytomegalovirus (CMV) promoter (described in U.S. Patent No. 4,168,062) and Rous Sarcoma virus promoter (described in Norton (1985) Molec. Cell Biol. 5:281).
  • CMV early cytomegalovirus
  • Rous Sarcoma virus promoter described in Norton (1985) Molec. Cell Biol. 5:281).
  • the desmin promoter (Li (1989) Gene 78:243; Li (1991) J. Biol. Chem. 266:6562; and Li (1993) J. Biol. Chem. 268:10401) is tissue specific and drives expression in muscle cells. More generally,
  • a composition of the invention can contain one or several nucleic acid molecules of the invention. It can also contain at least one additional nucleic acid molecule encoding another antigen or fragment derivative, including but not limited to, DPT vaccines, HMW protein of C. trachomatis or fragment thereof, MOMP of C. trachomatis or fragment thereof, entire organisms or subunits therefrom of Chlamydia, Neisseria, HIV Haemophilus influenzae, Moraxella catarrhalis, Human papilloma virus, Herpes simplex virus, Haemophilus ducreyi, Treponema pallidium, Candida albicans and Streptococcus pneumoniae, etc.
  • DPT vaccines HMW protein of C. trachomatis or fragment thereof
  • MOMP of C. trachomatis or fragment thereof entire organisms or subunits therefrom of Chlamydia, Neisseria, HIV Haemophilus influenzae, Moraxella catarrhalis, Human papill
  • a nucleic acid molecule encoding a cytokine, such as interleukin-1 or interleukin-12 can also be added to the composition so that the immune response is enhanced.
  • DNA molecules of the invention and/or additional DNA molecules may be on different plasmids or vectors in the same composition or can be carried in the same plasmid or vector.
  • nucleic acid molecules for therapeutic and prophylactic purposes include sterile saline or sterile buffered saline colloidal dispersion systems, such as macromolecule complexes, nanocapsules, silica microparticles, tungsten microparticles, gold microparticles, microspheres, beads and lipid based systems including oil-in-water emulsions, micelles, mixed micelles and liposomes.
  • a preferred colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (i.e., an artificial vesicle).
  • the uptake of naked nucleic acid molecules may be increased by incorporating the nucleic acid molecules into and/or onto biodegradable beads, which are efficiently transported into the cells. The preparation and use of such systems is well known in the art.
  • a nucleic acid molecule can be associated with agents that assist in cellular uptake. It can be formulated with a chemical agent that modifies the cellular permeability, such as bupivacaine (see, e.g., WO 9416737).
  • Cationic lipids are also known in the art and are commonly used for DNA delivery.
  • Such lipids include LipofectinTM also known as DOTMA (N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride), DOTAP (l,2-bis(oleyloxy)-3-(trimethylammonio)propane, DDAB
  • Cationic lipids for DNA delivery can be used in association with a neutral lipid such as DOPE (dioleyl phosphatidylethanolamine) as described in, e.g., WO 9011092.
  • DOPE dioleyl phosphatidylethanolamine
  • transfection facilitation compounds can be added to a formulation containing cationic liposomes. They include, e.g., spermine derivatives useful for facilitating the transport of DNA through the nuclear membrane (see, for example, WO 9318759) and membrane-permeabilizing compounds such as GAL4, Gramicidine S and cationic bile salts (see, for example, WO 9319768).
  • the amount of nucleic acid molecule to be used in a vaccine recipient depends, e.g., on the strength of the promoter used in the DNA construct, the immunogenicity of the expressed gene product, the mode of administration and type of formulation.
  • a therapeutically or prophylactically effective dose from about 1 ⁇ g to about 1 mg, preferably from about 10 ⁇ g to about 800 ⁇ g and more preferably from about 25 ⁇ g to about 250 ⁇ g can be administered to human adults.
  • the administration can be achieved in a single dose or repeated at intervals.
  • the route of administration can be any conventional route used in the vaccine field.
  • a nucleic acid molecule of the invention can be administered via a mucosal surface, e.g., an ocular, intranasal, pulmonary, oral, intestinal, rectal, vaginal, and urinary tract surface; or via a parenteral route, e.g., by an intravenous, subcutaneous, intraperitoneal, intradermal, intra-epidermal or intramuscular route.
  • a nucleic acid molecule formulated in association with bupivacaine is advantageously administered into muscles.
  • Recombinant bacterial vaccines genetically engineered for recombinant expression of nucleic acid molecules encoding an antigenic protein of the present invention include Shigella, Salmonella, Vibrio cholerae, and Lactobacillus.
  • Recombinant BCG and Streptococcus expressing one or more antigenic polypeptides can also be used for prevention or treatment of P. falciparum and/or P. yoelii infections.
  • Non-toxicogenic Vibrio cholerae mutant strains that are useful as a live oral vaccine are described in Mekalanos (1983) Nature 306:551 and U.S. Patent No. 4,882,278.
  • An effective vaccine dose of a Vibrio cholerae strain capable of expressing a polypeptide or polypeptide derivative encoded by a DNA molecule of the invention can be administered.
  • Attenuated Salmonella typhimurium strains genetically engineered for recombinant expression of heterologous antigens or not and their use as oral vaccines are described in Nakayama (1988) BioTechnology 6:693 and WO 9211361.
  • nucleic acid molecule(s) of the invention can be inserted into the bacterial genome, carried on a plasmid, or can remain in a free state.
  • nucleic acid molecules and polypeptides of the invention can be used sequentially or concomitantly as part of a multistep immunization process.
  • a mammal can be initially primed with a vaccine vector of the invention such as pox virus or adenovirus, e.g., via the parenteral route or mucosally and then boosted several time with a polypeptide e.g., via the mucosal route.
  • a mammal can be vaccinated with polypeptide via the mucosal route and at the same time or shortly thereafter, with a nucleic acid molecule via intramuscular route.
  • the antigenicity and/or immunogenicity of the peptides or fragments described herein may or may not necessarily require the use of an immunologically effective amount of an adjuvant or combination of adjuvants such as, but not limited to, alum, aluminum phosphate, aluminum hydroxide, squalene, oil-based adjuvants, virosomes, Q.S21, MF59, Army Liposomal Formulation (ALF) with or without QS-21 (Genito et al, Vaccine 35:3865 (2017)), interleukin 12 (IL-12), CpG, small molecule mast cell activator (MP7), TLR7 imidazoquinoline ligand 3M-019, resquimod (R848), N-acetyl-muramyl-L- threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (C
  • Table I provides information on adjuvants that may be useful. Table I shows possible adjuvants and their properties. These adjuvants may be used alone or in combination to test their ability to augment the immune response towards P. falcuparum and/or P. yoelii. These adjuvants are defined by their ability to drive a Thl or Th2 response.
  • Immunostimulatory agents or adjuvants have been used for many years to improve the host immune responses to, for example, vaccines.
  • Intrinsic adjuvants such as lipopolysaccharides, normally are the components of the killed or attenuated bacteria used as vaccines.
  • Extrinsic adjuvants are immunomodulators which are typically non-covalently linked to antigens and are formulated to enhance the host immune responses.
  • adjuvants have been identified that enhance the immune response to antigens delivered parenterally.
  • Aluminum hydroxide, aluminum oxide, and aluminum phosphate are routinely used as adjuvants in human and veterinary vaccines.
  • extrinsic adjuvants may include chemokines, cytokines (e.g., IL-2), saponins complexed to membrane protein antigens (immune stimulating complexes), pluronic polymers with mineral oil, killed mycobacteria in mineral oil, Freund's complete adjuvant, bacterial products, such as muramyl dipeptide (MDP) and lipopolysaccharide (LPS), as well as lipid A, and liposomes.
  • chemokines e.g., IL-2
  • saponins complexed to membrane protein antigens immunostimuls
  • pluronic polymers with mineral oil e.g., IL-2
  • MDP muramyl dipeptide
  • LPS lipopolysaccharide
  • mLT enterotoxigenic E. coli
  • U.S. Patent No. 4,855,283 which is incorporated herein by reference, teaches glycolipid analogues including N-glycosylamides, N-glycosylureas and N-glycosylcarbamates, each of which is substituted in the sugar residue by an amino acid, as immuno-modulators or adjuvants.
  • Lockhoff reported that N-glycosphospholipids and glycoglycerolipids are capable of eliciting strong immune responses in both herpes simplex virus vaccine and pseudorabies virus vaccine.
  • Some glycolipids have been synthesized from long chain-alkylamines and fatty acids that are linked directly with the sugars through the anomeric carbon atom, to mimic the functions of the naturally occurring lipid residues.
  • the immunogenic, antigenic, pharmaceutical, including vaccine, compositions may further comprise immune-effective amounts of an adjuvant, such as, but not limited to alum, mLT, LTR192G, Q.S21, RIBI DETOXTM, MM PL, CpG DNA, MF59, calcium phosphate, PLG interleukin 12 (IL12), TLR7 imidazoquinoline ligand 3M-019, resquimod (R848), small molecule mast cell activator MP7, ALF (with or without Q.S-21), and all those listed above.
  • an adjuvant such as, but not limited to alum, mLT, LTR192G, Q.S21, RIBI DETOXTM, MM PL, CpG DNA, MF59, calcium phosphate, PLG interleukin 12 (IL12), TLR7 imidazoquinoline ligand 3M-019, resquimod (R848), small molecule mast cell activator MP7, ALF (with or without
  • the adjuvant may be selected from one or more of the following: alum, Q.S21, CpG DNA, PLG, LT, 3D-mPL, or Bacille Calmette-Guerine (BCG) and mutated or modified forms of the above, particularly mLT and LTR192G.
  • the compositions of the present invention may also further comprise a suitable pharmaceutical carrier, including but not limited to saline, bicarbonate, dextrose or other aqueous solution. Other suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field, which is incorporated herein by reference in its entirety.
  • Immunogenic, antigenic and pharmaceutical, including vaccine, compositions may be administered in a suitable, nontoxic pharmaceutical carrier, may be comprised in microcapsules, microbeads, and/or may be comprised in a sustained release implant.
  • Table 2 provides a list of antigenic proteins useful in the methods and compositions of the present invention.
  • the "FC” indicates flow cytometry
  • "ES” indicactes ElisaSpot screening
  • P. yoelii indicates Plasmodium yoelli
  • P. falciparum indicates Plasmodium falciparum (isolate 3D7)
  • P. vivax indicates Plasmodium vivax (Sal-1).
  • RAS radiation-attenuated sporozoites
  • chloroquine hydrochloride Sigma-Aldrich
  • mice were immunized with 100 ⁇ g of DNA vector, pcDNA3.2-Dest (Invitrogen) in a 0.1 ml volume by intramuscular immunization. Six weeks later these mice were boosted with 1 x 10 10 pfu of Ad vector in a 0.1 ml volume. Both DNA and Ad were injected bilaterally into the tibialis anterior muscles with a 0.3 ml syringe and a 29.5 G needle (Becton Dickinson).
  • A20.2 J cells were grown in 15 ml of fresh RPMI-1640 media plus 20% FBS and 1% L- glutamine in 25 ml T-flasks. The T-flasks were kept upright and incubated in a 5 % CO2 incubator at 37°C overnight. When the cells reached a density of 1.2 -1.8 x 10 s cells/ml they were used to seed 12 well plates at a density of 5.0 x 10 5 cells/well. The following day the cells were infected with AdGFP, an adenovirus vector that expresses GFP, for 2 hours in a volume of 200 ⁇ . After infection, cells were washed with PBS, overlaid with 1 ml of fresh media and incubated at 37°C and 5% CC for 48 hours. The percentage of the GFP positive cells was analyzed by FACS.
  • A20.2J cells were infected with 200 ⁇ CPE lysate from each of the Ad- array vectors in 24 well plates for 2 hours. After infection, cells were washed with PBS, overlaid with 0.6 ml of fresh media and incubated at 37°C and 5% C0 2 for 24 hours.
  • splenocytes harvested from vaccinated animals were stimulated by co-culture with infected/irradiated A20.J2 cells in 96 well plates. Briefly, spleens were gently crushed using the flat end of a 3 cc or 10 cc syringe plunger, cell suspension was passed through a 70 ⁇ filter. The splenocytes were washed twice with 0.5 %FBS/10 mM HEPES/1 X HBSS. To remove the red blood cells ( BC), 5 ml of RBC lysing buffer (Sigma) were added to the cell pellets, and the tubes were swirled gently to mix the cells with the buffer, then incubated for 3 minutes at room temperature.
  • RBC lysing buffer Sigma
  • A20 cells were irradiated in a Pantak X-Rad 320 irradiator at 16,666 rads. After irradiation, 1.5xl0 5 infected cells were transferred to each well of U-bottom 96-well plates preloaded with 1x10 s splenocytes from vaccinated or naive mice, in triplicate, and incubated for 8 hours at 37°C. BD Golgi PlugTM (BD Bioscience) was added 1 hour into the incubation to block cytokine release. Cells were centrifuged at 1200 rpm for 5 minutes, the supernatant flicked, and the cell pellets resuspended by gentle vortexing.
  • Live and dead cells were first stained with Live/Dead Fixable Aqua stain kit (BD Biosciences), then the cells were blocked with FC BlockTM (BD Biosciences). After blocking, cell surface markers were stained with the following antibodies-(fluorochromes):CD4-eFlur-450 and CD8a-PerCP-Cy5.5 (BD Biosciences). Following separate fixation and permeabilzation steps, the samples were stained intracellularly with the following antibodies-(fluorchromes): IFNy,-PE, TNF-a, APC, and IL-2 -Alexa 488 (BD Biosciences).
  • FACSCaliburTM Becton Dickinson Immunocytometry Systems
  • AMS Automated Micro-sampling System
  • Transfection efficiency for each assay was monitored by transfection of the GFP-expressing control plasmid. Twenty-four hours post-transfection, cells were harvested and irradiated at 16,666 Rads, prior to plating for the IFN- ⁇ ELISpot assay. Multiscreen HTS HA 96-well filter plates (Millipore) were coated with 1 ⁇ g in 100 ⁇ of anti-mouse IFN- ⁇ antibody clone R4-6A2 in IX PBS pH 7.4. Plates were incubated overnight at room temperature, and then washed with RPMI.
  • Replication-incompetent adenovirus vectors contain a deletion in one or more replication- essential genes resulting in an adenovirus vector that cannot replicate in typical host cells, including a human patient.
  • a replication-incompetent adenovirus vector can be grown in a cell line that expresses the adenovirus genes necessary for replication.
  • replication-incompetent HuAd5 vectors that contain a deletion in the HuAd5 El region can be grown in the 293 cell line that expresses the HuAd5 El region
  • replication-incompetent HuAd5 vectors that contain a deletion in the HuAd5 El, E3 and E4 regions can be grown in the 2930RF6 cell line that expresses the HuAd5 El and E40RF6 regions.
  • Two different types of replication-incompetent HuAd5 vectors were used in the methods described herein: vectors that contain deletions in the El, E3 and E4 regions and vectors that contain a deletion in the El region.
  • Replication-incompetent HuAd5 E1-, partial E3-, E4- vectors were constructed using a method in which a foreign gene was recombined into a plasmid containing the HuAd5 genome in E. coli cells. Briefly, a Plasmodium gene was cloned into a small shuttle vector downstream from a human cytomegalovirus (HCMV) immediate-early (IE) promoter and between HuAd5 flanking arms.
  • HCMV human cytomegalovirus
  • IE immediate-early
  • the Plasmodium expression cassette was then recombined into a large plasmid containing the HuAd5 genome by transforming the small shuttle plasmid containing the Plasmodium expression cassette between HuAd5 flanking arms and a large plasmid containing the entire HuAd5 genome (minus HuAd5 El, E3 and E4 regions) into a recombination-positive strain of E. coli, BJDE3.
  • a recombinant plasmid in which the Plasmodium expression cassette has been recombined into the large HuAd5 plasmid was then identified by restriction enzyme analysis.
  • the large recombinant plasmid containing the Plasmodium expression cassette was then transformed into a recombination-negative strain of E. coli and isolated by standard microbiological methods.
  • the HuAd5 sequence (containing the Plasmodium expression cassette) was liberated from the large plasmid by digestion with a restriction endonuclease. This DNA was then transfected into 2930RF6 cells. Cell lysates were serially passaged every 3 - 4 days until cytopathic effect (CPE) was observed.
  • CPE is an indication that the viral vector is growing in the complementing cell line.
  • Virus was then expanded from a single 60 mm dish to at least 10 T175 flasks.
  • the recombinant vectors were released from infected cells by 3 freeze-thaws, treated with benzonase, purified by banding on a CsCI gradient, dialyzed with a HuAd5 buffer and stored at -80° C. Particle unit (pu) titers were then determined by absorbance at 260 nm.
  • Replication-incompetent HuAd5 El- vectors were generated using a site-specific recombination- based cloning method which allows for the transfer of DNA segments between different cloning vectors in vitro without the need for restriction endonucleases and ligase.
  • the GatewayTM cloning system relies on a site-specific recombination process between bacteriophage ⁇ and E. coli. Briefly, a Plasmodium gene was cloned into a kanamycin resistant (Kmr) GatewayTM "Entry" vector between two recombination sites (attLl and attL2).
  • the Plasmodium gene was then recombined into a large ampicillin resistant (Apr) GatewayTM "Destination" vector that contains the entire HuAd5 genome (minus the El region).
  • This "Destination" vector also contains two recombination sites (attRl and attR2) that flank a gene for negative selection, ccdB.
  • recombination sites attLl and attRl and between attL2 and attR2.
  • the product of this recombination event is a large plasmid in which the Plasmodium gene was cloned into the HuAd5 genome downstream from a HCMV IE promoter.
  • the large plasmid containing the Plasmodium expression cassette was then digested with a restriction endonuclease to liberate the HuAd5 sequence, and the DNA was transfected into 293 cells.
  • mice were immunized with a 100 ⁇ g of DNA vector expressing the specific antigen and then boosted 6 weeks later with an Ad5 vector (1 x 10 10 pfu) expressing the same antigen. Two weeks after the Ad5 boost, mice were challenged intravenously in the tail vein with 200 P. yoelii sporozoites using a 1 ml syringe and 26.5 G needle (Becton Dickinson).
  • Example 2 Generation of an Array of Adenovectors That Express a Panel of Highly Expressed P. yoelii Pre-erythrocytic Antigens
  • P. yoelii pre-erythrocytic genes with identifiable P. falciparum orthologs were selected for generation of an adenovector array (Ad-array) based on their level of expression in microarray and protein mass spectrometry datasets. Gene selection was made without regard to protein function or subcellular localization. In total, 312 P. yoelii genes were amplified from genomic DNA and cloned into El/E3-deleted adenovirus type 5 (Ad5) vector genomes ( Figure 2A). [00115] To facilitate high-throughput production of the Ad-array, the efficiency of adenovector generation was compared in multi-well plates of different sizes.
  • the adenovector plasmid had to convert into an adenovirus vector in sufficient quantities and quality to function in the antigen screening assay. Initially, conversions were tested of two pAd Flex plasmids that expressed the P. yoelii Hepl7 antigen (AdgHepl7) and the cytomegalovirus p65 antigen (AdgCMVp65). These large plasmids were transfected into 293 cells in 60-mm, 6-well, 12-well, 24-well, 48-well, and 96-well plates, and the cells were passaged to increase the adenovector titer.
  • AdgHepl7 the P. yoelii Hepl7 antigen
  • AdgCMVp65 cytomegalovirus p65 antigen
  • FIG. 3A The overall design of an antigen screening system is shown in Figure 3A. To test the elements of the screen, the MOI necessary to efficiently infect A20 cells was determined. Cells were infected with various doses of AdGFP, an Ad5 vector expressing GFP, and the percentage of infected cells was measured 48 hr post-infection (Figure 3B). MOI of 10, 100, or 1,000 focal forming units (ffus)/cell were required to infect approximately 2%, 10%, or 50% of the cells, respectively.
  • MHC histocompatibility complex
  • Ad-array vectors contain 25bp-long attB sequences flanking the transgene ( Figure 2B), which are remnants of the recombinase cloning reaction.
  • Ad-array vectors were directly compared with vaccine adenovectors, which do not carry the flanking attB sequences. The results indicate that the attB sequences did not inhibit the capacity to recall T cell responses in mice ( Figure 4C), indicating that Ad- array vectors are suitable for screening.
  • the 2,000 SPZ+CQ regimen was used to generate protective T cells for the identification of antigens.
  • Splenocytes were harvested 2 weeks after the last sporozoite immunization. The full array was screened simultaneously, in triplicate, against these freshly isolated splenocytes by ICS to identify pre- erythrocytic stage antigens able to recall IFNy-expressing CD8+ T cells.
  • A20 cells infected with 100 ffu/cell AdgPyCSP were included as a positive control.
  • Negative controls included uninfected A20 cells and A20 cells infected with 100 ffu/cell of AdNull and AdGFP vectors. The mean of the negative controls was 1% IFNy-expressing CD8+ T cells (Figure 5).
  • Antigens with responses greater than 2 SD of the mean of the negative controls were defined as positive hits in the screen.
  • 69 of the antigens in the array were positive and were targeted by CD8+ T cells induced in mice immunized with SPZ+CQ ( Figure 5).
  • CD4+ T cell responses and tumor necrosis factor (TNF)-a and interleukin (IL)-2 cytokines were analyzed by ICS.
  • CD4+ T cell responses were not observed in this system.
  • CD8+ TNF-a- expressing T cells were observed and tended to mirror the CD8+ IFNy responses. Very low levels of IL-2- expressing cells were observed.
  • Example 4 Identification of Protective Antigens [00122] Since the SPZ+CQ regimen induces protective T cell responses directed against antigens expressed in the pre-erythrocytic stages of the parasite life cycle, it was hypothesized that a subset of antigens identified in the SPZ+CQ screen would induce protective immune responses when delivered using a potent vaccine regimen designed to optimize CD8+ T cell responses.
  • the protective capacity of antigens was tested using a DNA prime-Ad5 boost regimen in BALB/c mice. Mice were immunized with 100 ⁇ g of DNA vector expressing the specific antigen and then boosted 6 weeks later with 1 ⁇ 10 10 particle units (PUs) of an Ad5 vector expressing the same antigen. Two weeks after the Ad5 boost, mice were challenged with P.
  • PUs 10 particle units
  • yoelii sporozoites and protection was monitored by microscopic examination of Giemsa-stained blood smears. Twenty-one percent (21%) of the PY00525 immunized mice were completely protected from sporozoite challenge, indicating that PY00525 can provide protection in mice. Twenty-one percent (21%) of the PY02793 immunized mice were completely protected from sporozoite challenge, indicating that PY02793 can provide protection in mice. Twenty-one percent (21%) of the PY03289 immunized mice were completely protected from sporozoite challenge, indicating that PY03289 can provide protection in mice.
  • mice Thirty-six percent (36%) of the PY03674 immunized mice were completely protected from sporozoite challenge, indicating that PY03674 can provide protection in mice.
  • the positive controls which were immunized with PyCSP expressing DNA and Ad5 vectors in the same regimen, protected 100% of the mice.
  • Example 5 The P. falciparum ortholog of PY03674 is immunogenic in BALB/c Mice.
  • falciparum ortholog of PY03674 was cloned into a highly immunogenic and low seroprevalent gorilla adenovector (GC46) and tested immunogenicity in mice.
  • PF3D7_0725100 (SEQ ID NO.: 17) was codon optimized for expression in mammals, synthesized, and used to produce GC46.PF3D7_0725100.
  • PF3D7_0725100 (1 ⁇ 10 9 PFU).
  • GC46.Null immunized and naive mice were included as control groups. At 21 days post-immunization, mice were euthanized for T cell studies. Antigen-specific T cell responses were measured from splenocytes by flow cytometry after stimulation with overlapping peptide pools and staining for cytokines and cell surface markers.
  • GC46. PF3D7_0725100 was immunogenic, inducing both antigen-specific CD8+ and CD4+ T cell responses ( Figure 6).
  • BALB/c mice were immunized with a single dose of 1 x 10 9 PFU of
  • mice were euthanized for splenocyte harvest and assessment of immune responses by ICS and flow cytometry. Splenocytes from
  • GC46.PF3D7_0725100 immunized mice were harvest and plated at 2 x 10 s cells per well in a 96 well v- bottom plate. Cells were stimulated for 4 hours in the presence of 20 ⁇ g/mL brefeldin A (Sigma-Aldrich) with either 15-mer peptides for the PF3D7_0725100 antigen at 2 ⁇ g/mL, overlapping by 10 amino acids (Mimotopes), or 1% DMSO as a negative control.
  • brefeldin A Sigma-Aldrich
  • CD4 Brilliant Violet 605 (clone RM4-5, Biolegend)
  • CD8 Horizon V500 (clone 53-6.7)
  • TNF Cy7PE (clone M P6-XT22)
  • IFNy allophycocyanin (clone XMG1.2)
  • IL-2 FITC clone JES6-5H4
  • flow cytometry was acquired by flow cytometry and cells were gated on forward scatter (threshold), exclusion of aggregates, and subsequently to include singlets, viable cells, CD14-, CD3+, CD19-, CD3+, lymphocytes, and either CD4+ or CD8+ populations.
  • Example 6 Identification of protective and immunogenic antigens using a matrix format
  • 20170232091A1 from 71% to 100% in both cases, demonstrating the ability of these antigens to work in combination with other vaccine antigens and enhance protective efficacy. This is important because multiple antigens may be combined to generate a successful subunit vaccine against P. falciparum and/or P. vivax malaria in humans.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Chemical & Material Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Organic Chemistry (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Immunology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne des procédés et des compositions pour immuniser un sujet contre le paludisme.
PCT/US2018/025510 2017-03-30 2018-03-30 Procédés et compositions pour vacciner contre le paludisme WO2018183922A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US16/497,244 US20210260176A1 (en) 2017-03-30 2018-03-30 Methods and Compositions for Vaccinating Against Malaria
EP18776071.5A EP3600399A4 (fr) 2017-03-30 2018-03-30 Procédés et compositions pour vacciner contre le paludisme

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201762479018P 2017-03-30 2017-03-30
US62/479,018 2017-03-30

Publications (1)

Publication Number Publication Date
WO2018183922A1 true WO2018183922A1 (fr) 2018-10-04

Family

ID=63676907

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2018/025510 WO2018183922A1 (fr) 2017-03-30 2018-03-30 Procédés et compositions pour vacciner contre le paludisme

Country Status (3)

Country Link
US (1) US20210260176A1 (fr)
EP (1) EP3600399A4 (fr)
WO (1) WO2018183922A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4147712A1 (fr) * 2021-09-13 2023-03-15 OncoDNA Procédé pour générer un groupe d'adn à double brin codant pour des néo-antigènes d'une tumeur d'un patient
WO2023036999A1 (fr) 2021-09-13 2023-03-16 Oncodna Procédé de génération d'un pool d'adn double brin codant pour des néo-antigènes d'une tumeur dun patient
EP4364752A1 (fr) 2022-11-07 2024-05-08 OncoDNA Vaccin ameliore

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022172264A1 (fr) * 2021-02-11 2022-08-18 Ramot At Tel Aviv University Ltd. Compositions et méthodes de traitement d'une maladie

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120014994A1 (en) * 2004-10-14 2012-01-19 Crucell Holland B. V. Malaria prime/boost vaccines
US20140010816A1 (en) * 2010-11-09 2014-01-09 Lin Chen Treatment and prevention of malaria

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080260763A1 (en) * 2004-07-01 2008-10-23 The Regents Of The University Of California High Throughput Proteomics
WO2007027860A2 (fr) * 2005-08-31 2007-03-08 Genvec, Inc. Vaccins contre la malaria a base de vecteurs adenoviraux
AP2008004448A0 (en) * 2005-09-30 2008-04-30 Seattle Biomedical Res Inst Plasmodium liver stage antigens
EP2451478A4 (fr) * 2009-11-05 2013-11-20 Us Navy Antigenes de sporozoites de plasmodium falciparum en phase hepatique

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120014994A1 (en) * 2004-10-14 2012-01-19 Crucell Holland B. V. Malaria prime/boost vaccines
US20140010816A1 (en) * 2010-11-09 2014-01-09 Lin Chen Treatment and prevention of malaria

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP3600399A4 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4147712A1 (fr) * 2021-09-13 2023-03-15 OncoDNA Procédé pour générer un groupe d'adn à double brin codant pour des néo-antigènes d'une tumeur d'un patient
WO2023036999A1 (fr) 2021-09-13 2023-03-16 Oncodna Procédé de génération d'un pool d'adn double brin codant pour des néo-antigènes d'une tumeur dun patient
EP4364752A1 (fr) 2022-11-07 2024-05-08 OncoDNA Vaccin ameliore
WO2024100025A1 (fr) 2022-11-07 2024-05-16 Oncodna Vaccin amélioré

Also Published As

Publication number Publication date
EP3600399A4 (fr) 2021-03-24
US20210260176A1 (en) 2021-08-26
EP3600399A1 (fr) 2020-02-05

Similar Documents

Publication Publication Date Title
Schneider et al. Enhanced immunogenicity for CD8+ T cell induction and complete protective efficacy of malaria DNA vaccination by boosting with modified vaccinia virus Ankara
US8012467B2 (en) Multivalent vaccines comprising recombinant viral vectors
AU2003297039B2 (en) Multi-antigenic alphavirus replicon particles and methods
JP4282095B2 (ja) Ｃｄ８ ｔ細胞免疫応答を発生するワクチン接種のための方法および試薬
EP1573012B1 (fr) Vaccins contre la malaria a base virale recombinante
US7981430B2 (en) Multi-clade chimeric immunogens derived from highly conserved regions of the human immunodeficiency virus type 1 (HIV-1) consensus proteome
US20210260176A1 (en) Methods and Compositions for Vaccinating Against Malaria
WO2008094188A2 (fr) Procédés et compositions utilisant la listeria pour renforcer l'immunogénicité par primo-immunisation
US11738073B2 (en) Immunogenic compositions, antigen screening methods, and methods of generating immune responses
Bruder et al. Molecular vaccines for malaria
WO1997039771A1 (fr) Vaccinations heterologues de rappel
US20050100558A1 (en) Heterologous boosting immunizations
WO2023118394A1 (fr) Vaccin à adn contre la leishmaniose
Han et al. 9 DNA Vaccines
Cell-Dependent et al. Recombinant Attenuated

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18776071

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2018776071

Country of ref document: EP

Effective date: 20191030